As disclosed in U.S. Pat. Nos. 5,888,505 and 6,294,168, eosinophil peroxidase may be used to selectively bind to and, in the presence of peroxide and halide, inhibit the growth of target microorganisms without eliminating desirable microorganisms or significantly damaging other components of the medium, such as host cells and normal flora, in the target microorganism's environment. Eosinophil peroxidase has previously been known to exhibit microorganism killing activity in natural systems when presented with an appropriate halide cofactor (X−) and hydrogen peroxide as substrate (Klebanoff, 1968, J. Bacteriol. 95:2131-2138). However, the selective nature of eosinophil peroxidase binding and the utility of these systems for therapeutic, research and industrial applications has only recently been recognized. Due to the newly discovered selective binding properties of eosinophil peroxidase, when a target microorganism, such as a pathogenic microorganism, has a binding capacity for eosinophil peroxidase greater than that of a desired microorganism, such as members of the normal flora, the target microorganism selectively binds the eosinophil peroxidase with little or no binding of the eosinophil peroxidase by the desired microorganism. In the presence of peroxide and halide, the target bound eosinophil peroxidase catalyzes halide oxidation and facilitates the disproportionation of peroxide to singlet molecular oxygen (1O2) at the surface of the target microorganism, resulting in selective killing of the target microorganism with a minimum of collateral damage to the desired microorganism or physiological medium. Thus, as disclosed in U.S. Pat. Nos. 5,888,505 and 6,294,168, eosinophil peroxidase can be employed as an antiseptic in the therapeutic or prophylactic treatment of human or animal subjects to selectively bind to and kill pathogenic microorganisms with a minimum of collateral damage to host cells and normal flora of the host.
The system may also be employed as disinfecting or sterilizing formulations for inhibiting the growth of target microorganisms in vitro, particularly in applications where biomedical devices, such as bandages, surgical instruments, suturing devices, catheters, dental appliances, contact lenses and the like, are antiseptically treated to inhibit the growth of target microorganisms without damage to host cells of a subject when the biomedical device is subsequently utilized in vivo.
As disclosed in U.S. Pat. Nos. 5,389,369 and 5,451,402, while the eosinophil peroxidase antiseptic system disclosed in U.S. Pat. Nos. 5,888,505 and 6,294,168 has been found to be highly effective in the treatment of pathogenic microbes, an antimicrobial activity enhancing agent may be required for the effective killing of yeast and spore forming microorganisms. The spore stage of the microbial life cycle is characterized by metabolic dormancy and resistance to environmental factors that would destroy the microbe in its vegetative stage. The earliest phase of spore germination is characterized by swelling and a shift from dormancy to active metabolism. Vegetative growth, e.g., sprouting, and ultimately reproduction follows.
Germination of bacterial endospores and fungal spores is associated with increased metabolism and decreased resistance to heat and chemical reactants. For germination to occur, the spore must sense that the environment is adequate to support vegetation and reproduction. The amino acid L-alanine is reported to stimulate bacterial spore germination (Hills, 1950, J Gen Microbiol 4:38; Halvorson and Church, 1957, Bacteriol Rev 21:112). L-Alanine and L-proline have also been reported to initiate fungal spore germination (Yanagita, 1957, Arch Mikrobiol 26:329).
Simple α-amino acids, such as glycine and L-alanine, occupy a central position in metabolism. Transamination or deamination of α-amino acids yields the glycogenic or ketogenic carbohydrates and the nitrogen needed for metabolism and growth. For example, transamination or deamination of L-alanine yields pyruvate which is the end product of glycolytic metabolism (Embden-Meyerhof-Parnas Pathway). Oxidation of pyruvate by pyruvate dehydrogenase complex yields acetyl-CoA, NADH, H+, and CO2. Acetyl-CoA is the initiator substrate for the tricarboxylic acid cycle (Kreb's Cycle) which in turns feeds the mitochondrial electron transport chain. Acetyl-CoA is also the ultimate carbon source for fatty acid synthesis as well as for sterol synthesis. Simple α-amino acids can provide the nitrogen, CO2, glycogenic and/or ketogenic equivalents required for germination and the metabolic activity that follows.
Accordingly, U.S. Pat. Nos. 5,389,369 and 5,451,402 disclose that the microbiocidal action of eosinophil peroxidase against yeast and sporular forms of microbes may be enhanced by treating the microorganisms with eosinophil peroxidase in combination with certain α-amino acids which provide a stimulating effect on yeast budding, germination of sporulated microbes, and possibly acceleration of metabolism of vegetative microbes. Representative α-amino acids disclosed for this purpose include glycine and the L- or D-enantiomers of alanine, valine, leucine, isoleucine, serine, threonine, lysine, phenylalanine, tyrosine, and the alkyl esters thereof. While U.S. Pat. Nos. 5,389,369 and 5,451,402 disclose the enhancement of microbiocidal activity of eosinophil peroxidase against yeast and sporular forms of microbes with α-amino acids, these patents do not disclose enhancement of the eosinophil peroxidase microbiocidal system against non-sporular bacterial or the further enhancement of antibacterial activity by the use of eosinophil peroxidase and at least two amino acids, as disclosed herein.